A Novel Intracellular K+/H+ Antiporter Related to Na+/H+ Antiporters Is Important for K+ Ion Homeostasis in Plants
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m210794200
ISSN1083-351X
AutoresKees Venema, Andrés Belver, M. Carmen Marín‐Manzano, María Pilar Rodríguez‐Rosales, Juan Pedro Donaire,
Tópico(s)Plant Molecular Biology Research
ResumoIn this study we have identified the first plant K+/H+ exchanger, LeNHX2 from tomato (Lycopersicon esculentum Mill. cv. Moneymaker), which is a member of the intracellular NHX exchanger protein family. The LeNHX2 protein, belonging to a subfamily of plant NHX proteins closely related to the yeast NHX1 protein, is abundant in roots and stems and is induced in leaves by short term salt or abscisic acid treatment. LeNHX2 complements the salt- and hygromycin-sensitive phenotype caused by NHX1 gene disruption in yeast, but affects accumulation of K+ and not Na+ in intracellular compartments. The LeNHX2 protein co-localizes with Prevacuolar and Golgi markers in a linear sucrose gradient in both yeast and plants. A histidine-tagged version of this protein could be purified and was shown to catalyze K+/H+ exchange but only minor Na+/H+ exchange in vitro. These data indicate that proper functioning of the endomembrane system relies on the regulation of K+ and H+ homeostasis by K+/H+ exchangers. In this study we have identified the first plant K+/H+ exchanger, LeNHX2 from tomato (Lycopersicon esculentum Mill. cv. Moneymaker), which is a member of the intracellular NHX exchanger protein family. The LeNHX2 protein, belonging to a subfamily of plant NHX proteins closely related to the yeast NHX1 protein, is abundant in roots and stems and is induced in leaves by short term salt or abscisic acid treatment. LeNHX2 complements the salt- and hygromycin-sensitive phenotype caused by NHX1 gene disruption in yeast, but affects accumulation of K+ and not Na+ in intracellular compartments. The LeNHX2 protein co-localizes with Prevacuolar and Golgi markers in a linear sucrose gradient in both yeast and plants. A histidine-tagged version of this protein could be purified and was shown to catalyze K+/H+ exchange but only minor Na+/H+ exchange in vitro. These data indicate that proper functioning of the endomembrane system relies on the regulation of K+ and H+ homeostasis by K+/H+ exchangers. With concentrations between 0.1 and 0.2 M, potassium is the most abundant cation in plant cells. The main pool of potassium inside the plant cell is in the vacuole. The function of potassium in this organelle is thought to be purely biophysical; to generate cell turgor to drive cell expansion (1Leigh R.A. Wyn Jones R.G. New Phytol. 1984; 97: 1-13Crossref Scopus (630) Google Scholar). Under conditions that limit K+ availability, the role of K+ in the vacuole can be replaced by other ions like Na+, as has been reported to occur under conditions of salt stress (2Binzel M.L. Hess F.D. Bressan R.A. Hasegawa P.M. Plant Physiol. 1988; 86: 607-614Crossref PubMed Google Scholar). Vacuolar concentrations thus vary from high (200 mM) to low (20 mM), suggesting the existence of active K+ import and export mechanisms at the vacuolar membrane (3Walker D.J. Leigh R.A. Miller A.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10510-10514Crossref PubMed Scopus (318) Google Scholar).The role and concentration of K+ in other endomembrane organelles in plants are largely unknown. Apart from a specific K+ requirement, secondary ion transporters might rely on K+ for pH regulation in these organelles. It was shown that apart from the vacuole, V-type H+-transporting ATPase is found in various membranes of the secretory system where vesicle acidification is important for ligand-receptor binding and protein modification, trafficking, and sorting (4Matsuoka K. Higuchi T. Maeshima M. Nakamura K. Plant Cell. 1997; 9: 533-546Crossref PubMed Google Scholar, 5Sze H. Li X. Palmgren M.G. Plant Cell. 1999; 11: 677-690PubMed Google Scholar). In animal cells, the pH gradient from neutral to acidic along organelles from both the secretory and endocytic pathways is suggested to be under tight control by the operation of secondary ion transporters providing proton leak pathways (6Schapiro F.B. Grinstein S. J. Biol. Chem. 2000; 275: 21025-21032Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 7Numata M. Orlowski J. J. Biol. Chem. 2001; 276: 17387-17394Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 8Grabe M. Oster G. J. Gen. Physiol. 2001; 117: 329-343Crossref PubMed Scopus (238) Google Scholar). Second, solute uptake energized by the pH gradient might be required to generate the osmotic pressure needed for vesicle fusion (5Sze H. Li X. Palmgren M.G. Plant Cell. 1999; 11: 677-690PubMed Google Scholar). In view of the abundance of K+ in the cell, K+/H+ exchangers are likely candidates to be involved in pH and osmoregulation of intracellular compartments as well as active uptake of K+ into vacuoles (3Walker D.J. Leigh R.A. Miller A.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10510-10514Crossref PubMed Scopus (318) Google Scholar) although the biochemical evidence for the existence of such antiporters is scarce (9Sarafían V. Poole R. Plant Physiol. 1987; 83: S255Google Scholar, 10Hassidim M. Braun Y. Lerner H.R. Reinhold L. Plant Physiol. 1990; 94: 1795-1801Crossref PubMed Scopus (73) Google Scholar).In contrast to the limited information available on K+/H+ antiporters, many reports have indicated the existence of vacuolar Na+/H+ antiporters in plants (11Blumwald E. Aharon G.S. Apse M.P. Biochim. Biophys. Acta. 2000; 1465: 140-151Crossref PubMed Scopus (760) Google Scholar). The first vacuolar Na+/H+ exchanger AtNHX1 was identified recently (12Gaxiola R.A. Rao R. Sherman A. Grifasi P. Alpier S.L. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1480-1485Crossref PubMed Scopus (509) Google Scholar), and it was shown that overexpression of this gene in plants enhances salt tolerance (13Apse M.P. Aharon G.S. Snedden W.A. Blumwald E. Science. 1999; 285: 1256-1258Crossref PubMed Scopus (1556) Google Scholar, 14Zhang H.X. Blumwald E. Nat. Biotechnol. 2001; 19: 765-768Crossref PubMed Scopus (870) Google Scholar, 15Zhang H.X. Hodson J.N. Williams J.P. Blumwald E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12832-12836Crossref PubMed Scopus (513) Google Scholar). A family of six genes was identified in Arabidopsis (AtNHX1 to AtNHX6) that shows sequence homology to mammalian and yeast NHE or NHX exchangers (16Yokoi S. Quintero F.J. Cubero B. Ruiz M.T. Bressan R.A. Hasegawa P.M. Pardo J.M. Plant J. 2002; 30: 529-539Crossref PubMed Scopus (427) Google Scholar, 17Nass R. Rao R. J. Biol. Chem. 1998; 273: 21054-21060Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). It was demonstrated however that AtNHX1 could catalyze both Na+/H+ and K+/H+ exchange (14Zhang H.X. Blumwald E. Nat. Biotechnol. 2001; 19: 765-768Crossref PubMed Scopus (870) Google Scholar, 18Venema K. Quintero F.J. Pardo J.M. Donaire J.P. J. Biol. Chem. 2002; 277: 2413-2418Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). A similar ion specificity was reported for the human NHE7 isoform. It was shown that this isoform is expressed in the trans-Golgi network, indicating that regulation of pH and ionic composition of intracellular compartments by K+/H+ or Na+/H+ exchange is an important task of these antiporters (7Numata M. Orlowski J. J. Biol. Chem. 2001; 276: 17387-17394Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). This notion was strengthened by the observation that the NHX1 protein is essential for osmotolerance and endosomal protein trafficking in yeast (19Nass R. Rao R. Microbiology. 1999; 145: 3221-3228Crossref PubMed Scopus (87) Google Scholar, 20Bowers K. Boaz P.L. Patel F.I. Stevens T.H. Mol. Biol. Cell. 2000; 11: 4277-4294Crossref PubMed Scopus (150) Google Scholar). Indeed, plant NHX genes were shown not only to be involved in salinity tolerance, but also in vacuolar pH regulation (21Fukada-Tanaka S. Inagaki Y. Yamaguchi T. Saito N. Iida S. Nature. 2000; 407: 581Crossref PubMed Scopus (132) Google Scholar, 22Yamaguchi T. Fukada-Tanaka S. Inagaki Y. Saito N. Yonekura-Sakakibara K. Tanaka Y. Kusumi T. Iida S. Plant Cell Physiol. 2001; 42: 451-461Crossref PubMed Scopus (150) Google Scholar), and to be induced by NaCl, KCl, and osmotic stress (12Gaxiola R.A. Rao R. Sherman A. Grifasi P. Alpier S.L. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1480-1485Crossref PubMed Scopus (509) Google Scholar, 16Yokoi S. Quintero F.J. Cubero B. Ruiz M.T. Bressan R.A. Hasegawa P.M. Pardo J.M. Plant J. 2002; 30: 529-539Crossref PubMed Scopus (427) Google Scholar, 23Quintero F.J. Blatt M.R. Pardo J.M. FEBS Lett. 2000; 471: 224-228Crossref PubMed Scopus (147) Google Scholar, 24Fukuda A. Nakamura A. Tanaka Y. Biochim. Biophys. Acta. 1999; 1446: 149-155Crossref PubMed Scopus (197) Google Scholar, 25Chauhan S. Forsthoefel N. Ran Y. Quigley F. Nelson D.E. Bohnert H.J. Plant J. 2000; 24: 511-522Crossref PubMed Google Scholar, 26Hamada A. Shono M. Xia T. Ohta M. Hayashi Y. Tanaka A. Hayakawa T. Plant Mol. Biol. 2001; 46: 35-42Crossref PubMed Scopus (161) Google Scholar), or even heat shock (27Porat R. Pavoncello D. Ben-Hayyim G. Lurie S. Plant Sci. 2002; 162: 957-963Crossref Scopus (31) Google Scholar).We have identified the first NHX proteins in tomato. One isoform, LeNHX2 is relatively distant from the AtNHX1 protein. In this study we describe this isoform, and analyze its function by heterologous expression in yeast. The LeNHX2 protein turns out to be the first intracellular K+/H+ antiporter protein in plants, which enables the maintenance of higher K+ concentrations in intracellular compartments under conditions of salt stress when expressed in yeast. These data show that plants contain different NHX genes with different ionic specificities regulating K+, Na+, and H+ ion homeostasis in intracellular compartments.EXPERIMENTAL PROCEDURESIsolation of LeNHX1 and LeNHX2—The Institute for Genomic Research (Rockville, MD) tomato EST data base (www.tigr.org) was searched in order to identify putative tomato genes with homology to NHE- or NHX-like proteins. Two ESTs (260098 and 254764) were identified and obtained from Clemson University Genomics Institute. Elongation of 5′- and 3′-ends was performed by nested PCR with two pairs of nested primers and a tomato (Lycopersicon esculentum Mill. cv. Moneymaker) root hair cDNA library (28Bucher M. Schroeer B. Willmitzer L. Riesmeier J.W. Plant Mol. Biol. 1997; 35: 497-508Crossref PubMed Scopus (58) Google Scholar) as template. One pair of primers was deduced from the λ polylinker sequence, and a pair of reversed gene-specific primers was derived from the EST sequences. Fragments were subcloned into pSTBlue-1, using Novagen's perfectly blunt cloning kit (Novagen, Madison, WI), and sequenced. The entire LeNHX2 coding sequence was amplified by nested PCR using a mixture of 7:1 Klenterm and Accuterm DNA polymerases (Labgen Molecular Biology) and cloned into the yeast expression vector pRS699, between the yeast PMA1 promoter and terminator (29Serrano R. Villalba J.M. Method. Cell Biol. 1995; 50: 481-496Crossref PubMed Scopus (25) Google Scholar), giving rise to plasmid pRS-LeNHX2. A different construct, containing a sequence coding for a RGSH10 epitope tag in the C-terminal end was amplified from the cDNA library and cloned in yeast expression vector pYes (Invitrogen), containing the GAL1 promoter, giving rise to plasmid pY-LeNHX2:H10. The coding region was sequenced in both directions to confirm the fidelity of the constructs. The complete cDNA sequences were deposited in the EMBL nucleotide sequence data base (LeNHX1 accession no. AJ306630, LeNHX2 accession no. AJ306631).Plant Growth Conditions—Tomato seeds (L. esculentum Mill, cv. Pera) were surface-sterilized and then germinated and grown for 5 weeks in sterile vermiculite under a light irradiance of 150 μmol m–2 s–1 (16-h photoperiod) at 26 °C and 60–65% relative humidity. One-tenth strength Hoagland's nutrient solution (30Hoagland D.R. Arnon D.I. Calif. Agricult. Exp. Stat. Circ. 1950; 347: 1-39Google Scholar) was applied from emergence of the first leaf and raised to quarter-strength (2 weeks after sowing) thereafter, every 3 days. Plants were treated for 0, 1, 6, and 24 h with 130 mm NaCl or for 1 h with 100 μm abscisic acid, before harvesting root, stem, and leaf tissues.RNA Blot Analysis—A DNA probe for LeNHX2 was made by amplification of a 415-bp fragment of the C-terminal coding region using gene-specific forward and reverse primers. Total RNA from different tissues was isolated and purified according to Logemann et al. (31Logemann J. Shell J. Willmitzer L. Anal. Biochem. 1987; 163: 16-20Crossref PubMed Scopus (1607) Google Scholar). RNA for all blots (15 μg) was run on 1.2% denaturing formaldehyde agarose gels and blotted onto nylon membranes (Hybond™ N+, Amersham Biosciences) (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning. A Laboratory Manual. 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 7.37-7.52Google Scholar). The DNA probes were radioactively labeled with [α-32P]dCTP by random priming using the Rediprime™II kit (Amersham Biosciences). Nylon filters were prehybridized and hybridized at 65 °C in hybridization buffer containing 7% (w/v) SDS, 300 mm sodium phosphate, pH 7.0, and 1 mm EDTA (33Church G.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7258) Google Scholar). Blots were washed twice in 4× SSC, 0.1 (w/v) SDS, and once in 0.4× SSC, 0.1 (w/v) SDS, at 65 °C for 15 min each. Membranes were rehybridized with a tomato 18 S rDNA gene probe to monitor RNA loading quantity (a gift from Dr. N. Ferrol, Est. Exp. Zaidín, CSIC, Granada, Spain). Nylon filters were exposed to a Phosphorimager screen (BioRad Molecular Imager System) and/or exposed to X-Omat AR5 film (Kodak), where hybridization signal was recorded with a Phosphorimager analyzer (BioRad Molecular Imager System) or digitized with a BioRad imaging analyzer, respectively. Transcript amount was determined by quantification of band signal intensities of digitized images and relativized to the respective ribosomal signals using Scion Image software (version 3.62a; www.scioncorp.com).Yeast Strains and Growth Conditions—All Saccharomyces cerevisiae strains used were derivatives of W303-1B (Matα leu2-13 112 ura3-1 trp1-1 his3-11, 15 ade2-1 can1-100). Strains WX1 (Δnhx1::TRP1), ANT3 (Δena1–4::HIS3 Δnha1::LEU2), and AXT3 (Δena1–4::HIS3 Δnha1::LEU2 Δnhx1::TRP1) were gifts from Drs. F. J. Quintero and J. M. Pardo (IRNA Sevilla, Spain). Cells were grown at 30 °C in YPD (1% yeast extract, 2% peptone, 2% glucose) or APD (10 mm arginine, 8 mm phosphoric acid, 2% glucose, 2 mm MgSO4, 1 mm KCl, 0.2 mm CaCl2, trace minerals and vitamins, Ref. 34Rodríguez-Navarro A. Ramos J. J. Bacteriol. 1984; 159: 940-945Crossref PubMed Google Scholar). For induction of antiporter expression in cells carrying plasmids with antiporter genes under the control of the GAL1 promoter, glucose (2%) was replaced by galactose (2%) in the growth media. For growth curves or drop tests, a preculture was made by growing the strains in selective APD medium to saturation (OD660 nm of 4–5). In drop tests, the preculture was diluted 50-fold, after which 10 μl of serial (10–1) dilutions were spotted on YPD plates with 0, 10, or 25 μg/ml hygromycin B. Growth curves were made in a total volume of 50 ml using a 250-ml cell culture vessels stirred at 80 rpm under continuous aeration. Growth was started by diluting the preculture to OD660 nm of 0.006 in APD medium (pH 6.0) with or without 20 mm NaCl. Samples were taken at the indicated time points to determine OD660 nm.Determination of Total Intracellular Ion Content—Cells were grown in liquid APG medium (APD medium with galactose instead of glucose) supplemented or not with 20 mm NaCl as indicated. 50-ml fractions were taken when the cells reached an OD660 nm of 0.25 ± 0.01 and centrifuged (2 min at 3000 × g). Cells were washed twice with 10 ml of ice-cold 10 mm MgCl2, 10 mm CaCl2, 1 mm HEPES. Washed cells were resuspended in 2 ml of the same buffer and the cell density determined from the optical density at 660 nm. Separately the relationship between OD660 nm and yeast dry weight was determined. Intracellular ions were extracted by addition of HCl to a final concentration of 0.4% and incubation for 20 min at 95 °C. After removal of cell debris by centrifugation, potassium and sodium ion content was determined with an atomic absorption spectrometer.Determination of Vacuolar and Cytoplasmic Ion Content—The vacuolar and cytoplasmic Na+ and K+ content was determined by treating the cells with cytochrome c that selectively permeabilizes the plasma membrane (35Okorokov L.A. Lichko L.P. Kulaev I.S. J. Bacteriol. 1980; 144: 661-665Crossref PubMed Google Scholar, 36Ramos J. Haro R. Rodríguez-Navarro A. Biochim. Biophys. Acta. 1990; 1029: 211-217Crossref PubMed Scopus (75) Google Scholar). Cells were grown and washed as above and resuspended in 50 μl of 2% cytochrome c, 18 μg/ml antimycin, 1 mm HEPES, 10 mm MgSO4, 10 mm CaCl2 and 5 mm 2-deoxy d-glucose. After incubation for 20 min at room temperature, the cells were centrifuged and washed three times with 2 ml of the same solution without cytochrome c. The supernatants containing the ions released by the cytochrome c treatment were pooled to determine cytoplasmic ion content. The remaining ions, corresponding to the vacuolar fraction, were extracted by addition of HCl to a final concentration of 0.4% in a total volume of 2 ml and incubation for 20 min at 95 °C. Potassium and sodium ion content was determined with an atomic absorption spectrometer as above.Yeast Membrane Fractionation—Microsomal membranes were isolated as described (18Venema K. Quintero F.J. Pardo J.M. Donaire J.P. J. Biol. Chem. 2002; 277: 2413-2418Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) The microsomal membrane pellet (2 ml) was layered on a linear (28 ml) 20–55% sucrose gradient prepared in 10 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1 mm dithiothreitol, and 30 μl of protease inhibitor mixture (Sigma), and centrifuged 18 h at 100,000 × g in a Beckman SW28 swinging bucket rotor. 2-ml fractions were collected, and sucrose density and protein content determined.Tomato Cell Membrane Fractionation—Microsomal membranes were obtained from Tomato calli (L. esculentum Mill. cv. Pera) as previously described (37Rodríguez.-Rosales M.P. Kerkeb L. Bueno P. Donaire J.P. Plant Sci. 1999; 143: 143-150Crossref Scopus (75) Google Scholar). Microsomal membranes were layered on a linear (14 ml) 20–40% sucrose gradient and centrifuged as described above. 1-ml fractions were collected, and sucrose density and protein content determined.Marker Enzyme Analysis—In tomato cell membrane fractions latent UDPase activity (Golgi) (38Green J.R. Hall J.L. More A.L. Isolation of Membranes and Organelles from Plant Cells. Academic Press, London1983: 135-152Google Scholar) Antimycin A-insensitive NADH-cytochrome c reductase (ER) 1The abbreviations used are: ER, endoplasmic reticulum; Mes, 4-morpholineethanesulfonic acid; BTP, 1,3-bis[tris(hydroxylmethyl)-methylamino]propane; EIPA, ethylisopropyl-amiloride. (39Hodges T.K. Leonard R.T. Methods Enzymol. 1974; 32: 392-406Crossref PubMed Scopus (576) Google Scholar) and nitrate-sensitive ATPase (Tonoplast) (40O′Neil S.D. Bennet A.B. Spanswick R.M. Plant Physiol. 1983; 72: 837-846Crossref PubMed Google Scholar) was measured. The LeNHX2 protein was detected by immunoblot analysis using the affinity-purified polyclonal antibody raised against the LeNHX2 c-terminal peptide EPIMHSSRRAGYDGH (Sigma-Genosys). In yeast membrane fractions marker enzyme proteins were detected by immunoblot analysis. The distribution of ER membranes was assayed by determination of NADPH-cytochrome c oxidoreductase activity (41Schatz G. Klima J. Biochim. Biophys. Acta. 1964; 81: 448-461PubMed Google Scholar). SDS-PAGE and Western blotting were performed as described (18Venema K. Quintero F.J. Pardo J.M. Donaire J.P. J. Biol. Chem. 2002; 277: 2413-2418Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) using a monoclonal antibody raised against the 100-kDa subunit of the vacuolar proton ATPase VPH1, a monoclonal antibody against the late Golgi protein vps10p, and a monoclonal antibody against the endosomal/prevacuolar protein pep12p (Molecular Probes). The recombinant LeNHX2 protein in yeast was detected using a monoclonal antibody raised against the RGSH4 epitope (Qiagen).Purification of LeNHX2:H10 Protein by Ni2+ Affinity Chromatography—The LeNHX2 protein was purified essentially as described for the AtNHX1 protein (18Venema K. Quintero F.J. Pardo J.M. Donaire J.P. J. Biol. Chem. 2002; 277: 2413-2418Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) with some modifications. Cells were grown in APG medium to an OD660 nm of 1.0. Microsomal membranes were isolated as described (18Venema K. Quintero F.J. Pardo J.M. Donaire J.P. J. Biol. Chem. 2002; 277: 2413-2418Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) after which the microsomal membrane fraction (4 ml, 10 mg of protein/ml in 100 mm Tris-HCl, pH 7.5, 20% glycerol, 0.1 mm dithiothreitol, and 0.1 mm EDTA) was mixed with 40 ml of solubilization buffer (50 mm KH2PO4, pH 7.4, 500 mm NaCl, 10 mm imidazole, 20% glycerol, 0.5% n-dodecyl-β-d-maltoside) supplemented with 200 μl of protease inhibitor mixture (Sigma) and incubated for 30 min at 4 °C under gentle shaking. Unsolubilized material was removed by centrifugation for 30 min at 30,000 × g (Sorvall SS34 rotor). The supernatant was mixed with 1 ml of Ni-nitrilotriacetic acid; resin (Qiagen) and incubated overnight at 4 °C. The resin was then poured into a polypropylene column and prewashed with an imidazole step-gradient of 8-ml fractions containing 20, 50, 75, and 100 mm imidazole (pH 7.4) in 50 mm KH2PO4 (pH 7.4), 500 mm NaCl, 10% glycerol, 0.075% n-dodecyl-β-d-maltoside, 2 μg/ml pepstatin, 0.2 mm phenylmethylsulfonyl fluoride. Finally, bound protein was washed with 4 ml of 20 mm BTP-Mes (pH 7.4), 100 mm imidazole, 10% glycerol, 0.075% n-dodecyl-β-d-maltoside, 2 μg/ml pepstatin, and 0.2 mm phenylmethylsulfonyl fluoride and eluted slowly in 10 × 0.5 ml of 20 mm BTP-Mes pH 7.4, 500 mm imidazole (pH 7.4), 10% glycerol supplemented with 2 μg/ml pepstatin, and 0.2 mm phenylmethylsulfonyl fluoride. The third fraction would normally contain 50–75 μg of purified antiporter protein, corresponding to 65% of totally eluted antiporter protein. This fraction was frozen in liquid nitrogen and stored at –80 °C, until use for subsequent experiments.Reconstitution of LeNHX2:H10 Protein and Measurement of Cation/H+ Exchange in Vitro—Reconstitution was performed by elimination of detergent using Sephadex G-50 (fine; Amersham Biosciences) spin columns, and Biobeads (SM-2; Bio-Rad) as previously described (18Venema K. Quintero F.J. Pardo J.M. Donaire J.P. J. Biol. Chem. 2002; 277: 2413-2418Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) using 5 μg of protein and in the presence of ammonium sulfate. In order to encapsulate pyranine during the reconstitution, it was essential to preload the G-50 spin column with 200 μl of 2.5 mm pyranine (BTP salt) in reconstitution buffer, as the pyranine present in the liposome-protein-detergent mixture was eliminated by >99% at the top of the column. This shows that the reconstitution takes place along the passage of the sample through the column, allowing also an efficient elimination of imidazole or traces of KCl and NaCl in the protein sample. An inside acid pH gradient was created by diluting the proteoliposomes 50-fold in (NH4)2SO4-free medium. Cation/H+ exchange activity was monitored by measuring the rise in fluorescence of encapsulated pyranine in the presence of various monovalent cations as previously described (18Venema K. Quintero F.J. Pardo J.M. Donaire J.P. J. Biol. Chem. 2002; 277: 2413-2418Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). For measurement of antiporter activity in the absence of a preimposed pH gradient, ammonium sulfate in the reconstitution was replaced by Cs2SO4, pH during reconstitution was adjusted to 7.2, and measurements were made in the same buffer containing equilibrium Cs2SO4 concentration.Affinity Purification of the LeNHX2 Polyclonal Antibody—A rabbit polyclonal antibody was raised against a peptide corresponding to the C-terminal 15 amino acids of the LeNHX2 protein (Sigma-Genosys). The antibody was purified from the serum by affinity purification using the LeNHX2 protein. 10 μg of purified protein was separated by SDS-gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The band containing the protein was cut from the membrane, blocked, and incubated for 3 h with the serum at a 1:20 dilution in 1.5 ml of 20 mm K-phosphate, pH 7.5, 150 mm NaCl, 0.1% bovine serum albumin. After washing, the antibody was eluted with 750 μl of 0.2 m glycine, pH 2.7, 1 mm EGTA and neutralized with an equal volume of 0.2 m Tris-HCl, pH 8.0, 0.2% bovine serum albumin, 0.02% NaN3. The purified antibody solution was used at a 1:10 dilution in Western blot experiments.Protein Determination—Protein was determined by the method of Schaffner and Weissmann (42Schaffner W. Weissmann C. Anal. Biochem. 1973; 56: 502-514Crossref PubMed Scopus (1946) Google Scholar) with bovine serum albumin as standard.RESULTSIsolation and Molecular Description of LeNHX1 and LeNHX2—To clone antiporters of the NHX family from tomato we aimed at identifying homologues of the Arabidopsis thaliana vacuolar (Na+,K+)/H+ antiporter AtNHX1. Two ESTs were identified and the complete corresponding cDNAs were obtained based on PCR elongation of 3′- and 5′-coding sequences. The open reading frames of LeNHX1 and LeNHX2 encode for proteins of 531 and 534 amino acids, respectively. Both predicted proteins, which are only 31% identical, are closely related to the family of intracellular Na+/H+ antiporters found in animals, plants, and fungi (Figs. 1 and 2) (16Yokoi S. Quintero F.J. Cubero B. Ruiz M.T. Bressan R.A. Hasegawa P.M. Pardo J.M. Plant J. 2002; 30: 529-539Crossref PubMed Scopus (427) Google Scholar, 17Nass R. Rao R. J. Biol. Chem. 1998; 273: 21054-21060Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 43Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1988; 273: 6851-6959Google Scholar, 44Nehrke K. Melvin J.E. J. Biol. Chem. 2002; 277: 29036-29044Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). In Arabidopsis, 6 members of this family were identified that could be subdivided into 2 relatively distant groups (16Yokoi S. Quintero F.J. Cubero B. Ruiz M.T. Bressan R.A. Hasegawa P.M. Pardo J.M. Plant J. 2002; 30: 529-539Crossref PubMed Scopus (427) Google Scholar). All NHX proteins described from other plant species fall within the same group as the AtNHX1 protein constituting a group of closely related plant NHX sequences. As the proteins from the second group are much less known, we focused this study on a description of the functioning of the LeNHX2 protein.Fig. 2Comparison of the primary structure of NHX sequences from different organisms. The amino acid sequence of NHX proteins representative of the different groups shown in Fig. 1 were aligned using ClustalX 1.5b. Positions containing conserved amino acids are shaded black, while positions containing identical amino acids in all sequences are marked with an asterisk. The membrane topology of the LeNHX1 and LeNHX2 proteins, as predicted at genome.cbs.dtu.dk (56Sonnhammer E.L.L. von Heijne G. Krogh A. Glasgow J. Littlejohn T. Major F. Lathrop R. Sankoff D. Sensen C. Proc. Sixth Int. Conf. On Intelligent Systems for Molecular Biology. AAA1 Press, Menlo Park, CA1998: 175-182Google Scholar) is shown above the sequence by a filled (transmembrane segment for both proteins) or open (transmembrane segment for one of the two proteins) bar. Also indicated are segments 6 and 7 that were slightly below the threshold in this prediction for both proteins. The numbering of the transmembrane segments is based on the topology prediction for the human NHE1 protein (57Wakabayashi S. Pang T. Su X. Shigekawa M. J. Biol. Chem. 2000; 275: 7942-7949Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The first transmembrane segment is absent from all the plant sequences, while the transmembrane segment between TM9 and TM10 (not numbered) is proposed to be extracellular in the NHE1 model. Amino acids in the human NHE1 isoform that have been shown to affect the inhibition of antiporter activity by amiloride derivatives (52Counillon L. Franchi A. Pouysségur J. Proc. Natl. Acad. Sci. U. S. 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Chem. 1997; 272: 26145-26152Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). To test the involvement of the LeNHX2 protein in these processes we subjected tomato plants of 5-week-old grown in vermiculite to a shock of 130 mm NaCl. The LeNHX2 g
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